Kamis, 25 Agustus 2011

Human cytomegalovirus (CMV) can cause severe, life-threatening disease in immunocompromised patients such as transplant patients and patients with AIDS. Systemic infections are characterized by carriage of CMV in the polymorphonuclear leukocytes (PMNL) of peripheral blood (viremia). Infected PMNL can be detected by direct detection of CMV pp 65 antigen (CMV antigenemia) using an indirect immunofluorescence (IFA) technique. CMV antigenemia can also be used to monitor the course of CMV infection during and after treatment. Antigenemia will be performed on EDTA or heparinized blood requesting CMV. Antigenemia and culture will be performed on bone marrow samples requesting CMV.

II. Collection and Transport

A minimum of 5 mL of blood is collected in an EDTA Vacutainer® tube (purple top). Samples should be transported to the laboratory as soon as possible at room temperature. Smaller volumes of blood from infants will be accepted and the procedure will be completed despite a small number of PMNL’s. Blood samples received 14:00–13:00 hrs will be processed as far as preparation and fixing of slides. Staining and reading will be done the next day. Samples received after 15:00 hrs will be refrigerated and processed the next working day or a fresh sample requested (except on Fridays, consult Charge technologist).

Note:

Toronto Hospital Division of UHN: Reject (Transplant patients, In or Out patients)

1. Specimens received >24 hours after being taken.

2. Specimens received over the weekend (Friday after 2 p.m. to Sunday midnight)

Other sites (PMH, MSH): Only reject specimens if received over the weekend (i.e. >24 hours after collection.)

In-house positive CMV control (stored at -20oC and prepared from positivepatients cells).Use with first batch of slides being stained each day.

In-house positive CMV control (stored at 4oC and prepared from positiveCMV cultures).These are double-well slide with CMV positive and negative wells.Use 1 control with each subsequent batch being stained.

Commercially prepared CMV control (stored at 4oC) Use with each new batch of PP65 that is prepared(or if there are no other control slides available)

Gently pour supernatant into a discard container. Carefully remove excess red blood cells from suspension above the WBC deposit.(fluff the cells by using the pipette to gently blow the RBC’s off the button).

Gradually add PBS to cell pellet until it reaches a turbidity of ~ 1.0 to 2.0 MacFarland Standard.

· Run the Isotonic Diluent 3 times using the Start/Stop key. (Record with check marks on the daily scheduled maintenance section on Equipment sheet Policy QEQMI103001hCoulter.01) in the black binder titled “Coulter Analyser History Log”. (Record with check marks on the daily scheduled maintenance section on Equipment sheet Policy QEQMI103001hCoulter.01) in the black binder titled “Coulter Analyser History Log”.

· On Mondays (or Tuesdays following a long weekend) run the CBC commercial control for Coulter Particle Counter (STRECK Para 4) as follows:

a. Remove vials from the refrigerator and allow to warm up to room temperature. (18-30C.)

b. Date the vial when it is used for the first time.

c. Use the red topped vial. (It is the lower control.)

d. Mix the vial by holding it horizontally between the palms of the hands. (Do NOT use a mechanical mixer). Roll the vial back and forth for 20-30 seconds; occasionally invert the vial. Mix well (to resuspend the red cells, but don not shake.

e. Gently invert the vial 8-10 times immediately before sampling.

f. Pipette 50ul of sample from the red topped vial into 10 mL of Isoton diluent.

g. Add 3 drops of Zap-oglobin and mix well.

h. Using the Coulter Particle Counter, count this sample 3 times. Record the counts in the Coulter Particle Counter Log Sheet found in the “Coulter Analyser History Log” black binder. Note: there is a separate log sheet for each kits lot number. Start a new sheet for each subsequent lot number. Also put a check mark in the appropriate box of the weekly scheduled maintenance sheet Policy QEQMI03001hCoulter.01. (also found in the black binder.) Put the manufacturers parameter sheet for each new lot number in the binder and put the date first used on the sheet.

Dispense 10 mL of Isotonic Solution into an ACCUVETTE II vial for each sample.

Invert cell suspension; dispense 50 uL of cell suspension into each ACCUVETTE II vial. Pipette up and down several times.

Add 3 drops of ZAP-OGLOBIN (which will lyse any remaining RBC’s).

Stir with transfer pipette to mix. Avoid introducing air bubbles.

Read immediately two times and calculate the average WBC count (e.g. Reading of 2,200 E6 means the cell suspension has a WBC count of 2.2 x 106 /mL). Write down the average WBC count on the centrifuge tube.

Acceptable limit is between 1.0 x 106 to 2.0 x 106/mL

Increase the volume added to the cytospin funnel if a specimen has a very low WBC count (maximum 300 uL). Each slide should have 200, 000 WBC’s.

NOTE: If you get a count just under 1.0 x 106, you could write x2 on the lid of the conical tube and double the amount added to the cytospin funnel.) If you do this, you must also double the count i.e. an initial count of 0.9 x 106/ml would be calculated as 1.8 x 106 /mL.

At the end of the day:

· Immerse the Aperature Tube of the Coulter Counter into an ACCUVETTE II vial full of blue Cleaner Solution.

5. Let slides dry in Laminar Flow Hood for 5 minutes and proceed to staining, or store dried slides in fridge at 4oC for up to 72 hr. Store in -70oC freezer if slides cannot be stained within 72 hr.

E. Staining of Slides

1. Place one double-well control slide with CMV positive and negative cell spots at a random position within each batch of slides to be stained.

Add 20 ml of working solution monoclonal antibody # 1 (anti-pp65) onto the sample and control slide. Incubate in a humidified chamber at 37°C for 30 minutes. FROM THIS POINT, DO NOT ALLOW THE CYTOPREP TO DRY AT ANY TIME DURING THE STAINING PROCESS.

Wash by immersion in fresh PBS 3 times for 1 minute each.

Wipe excess PBS off slide and add 20 ml of fluorescein conjugated antibody # 2 to the cytoprep. DO THIS ONE SLIDE AT A TIME, DO NOT ALLOW CELL SPOT TO DRY IN BETWEEN.

a. Check reagent expiratory date and verify that Reagent QC is satisfactory for the reagent lot/kit being used

b. Appropriate control slide with positive and negative CMV wells (commercial; home-made slide with ATCC strain or buffer coat of known CMV positive are acceptable) must be stained with each batch. Place QC slide at a random position within each batch

c. Examine the negative control well first to establish the dull red colour (Evans blue counterstain) and to determine if there is any nonspecific staining.

The positive control must be clearly distinguishable from the negative control or the test is invalid.

e. If the re-run shows the old QC material still fails, fresh QC passes and nothing else is wrong with the batch (only the old QC material failed, patient results valid) patient results may be released.